PROJECT SUMMARY The heart is a highly complex biological system. The overall goal of this project is to use multiscale computational modeling of the heart from the molecular level to the organ level to identify the pro-arrhythmic effects of structural and functional heterogeneity and elucidate molecular and ionic mechanisms of calcium (Ca2+) waves, delayed afterdepolarizations (DADs), premature ventricular contractions (PVCs), and thus ventricular fibrillation (VF). A key outcome will be to provide physiological bases for antiarrhythmic drug development, gene therapies, and novel therapeutic strategies. The project builds on our recent discoveries 1) heterogeneous cell-to-cell coupling promotes triggered arrhythmias at the tissue scale; 2) heterogeneous ryanodine receptor (RyR) distribution promotes arrhythmogenic Ca2+ sparks and waves at the subcellular scale. The work proposed here is aimed at bridging the knowledge gap between the tissue scale arrhythmia mechanisms and the subcellular scale arrhythmia mechanisms utilizing multiscale computational modeling and the state-of-the-art experimental approaches to measure detailed heterogeneity in the heart. Aim #1 is to establish link between RyR properties and subcellular Ca2+ dynamics. To do this, we will extend this study and investigate heart failure (HF) cells, which are supposed to be more heterogeneous. We will measure RyR distributions in normal and HF cells and build the physiological and pathological models to test our hypothesis that heterogeneous RyR distribution promotes Ca2+ waves, DADs, PVCs, and thus focal arrhythmias. Key questions that we will address in Aim #1 are: 1) how RyR cluster size and spatial arrangements of RyRs at the cleft space affect Ca2+ sparks; 2) how RyR cluster distribution in the cell promotes arrhythmogenic Ca2+ waves. RyR gating, and thus Ca2+ sparks and waves, are also influenced by posttranslational modifications (PTMs). Aim #2 is to test the hypothesis that PTMs further increase heterogeneous Ca2+ transients interacting with structural RyR heterogeneity. SERCA reuptake is another key player in the Ca2+ cycling. Increasing SERCA pump activity increases SR Ca2+ load, which promotes wave propagation. At the same time, increasing SERCA pump activity reduces cytosolic Ca2+ transients, which suppresses wave propagation. In Aim #3, we test the hypothesis that increasing SERCA-pump function has a biphasic effect on propensity of arrhythmogenic Ca2+ waves. When Ca2+ waves occur, they depolarize the cell membrane and can lead to triggered activity in tissue. If cells are well-coupled, depolarization will be immediately absorbed by surrounding cells. However, when cell-to-cell coupling is reduced, depolarization cannot be absorbed by surrounding cells and PVCs occur more easily. However, at the same time, reduced cell-to-cell coupling makes wave propagation more difficult. Therefore, we hypothesize that there is an optimal cell-to-cell coupling for PVC formation (Aim #4). The proposed work will establish a new paradigm that a few irregular Ca2+ sparks can lead to the whole heart arrhythmias when cardiac heterogeneity is increased in HF and other pathological conditions.